Biology subjects

Loh, Y. H. E.

Publications and source records attributed to Loh, Y. H. E..

5 recordsLinked to original sources

bioRxiv2026

Mutation count and mutation profile analyses of single-nucleotide variants in single human colon crypts

Genetic heterogeneity due to the accumulation of mutations in normal tissues can increase cancer risk and be an important factor in many age-related degenerative and chronic diseases. Somatic mutations can arise from DNA damage or replication errors and accumulate in normal tissues with age. We obtained high depth whole genome sequencing data to comprehensively profile somatic mutations in 106 single human colon crypts with matched bulk controls from 21 individuals age 10 months to 90 years old. Our analysis reveals that about half of the human crypts are polyclonal (multi-lineage) instead of entirely monoclonal as conventionally construed. Consequently, the DNA mutation count would be inflated, while the variant allele frequency for each variant would be reduced in the cell population of a multi-lineage colon crypt. Therefore, our mutation count analysis includes using single stem cell lineage colon crypts exclusively to establish the somatic mutation rate for each of the 96 trinucleotide mutation categories. In addition, the mutation profile, representing the relative presence of the 96 trinucleotide mutation categories, in each colon crypt is analyzed. Unlike mutation count, the mutation profile is not affected by crypt clonality and is very similar across all ages in individuals with no chemotherapy or radiation treatment. Importantly, combined results from mutation count and mutation profile analyses of individuals with chemotherapy or radiation suggest an intriguing impact of these treatments on cell survival. The baseline mutation rate and the normal mutation profile established in our study provide a framework for a genomic standard to assess biological age and deviations associated with factors such as lifestyle, age-related degenerative and chronic diseases, and potentially cancer treatment outcome. Future studies similar to this current study on other tissues can provide further insight into how and why different tissues age differently in humans.

genomics↗

bioRxiv2026

Complex Indel Detection: A Simulation-Based Framework and Parsing with FreeBayes

In contrast to simple deletions and simple insertions, most complex indels involve both deletions and insertions, often with base changes within a few nucleotides of the indels left and right boundaries. These complex indels often arise from double-strand breaks (DSB), which in normal somatic cells are predominantly repaired by nonhomologous DNA end joining (NHEJ). Such complex indels pose a difficult analytical problem for existing indel callers because the observed VCF representation may be locally shifted, extended with matching flanking bases, or fragmented into several closely spaced calls. To evaluate complex indel representation, we tested six variant calling approaches: FreeBayes, HaplotypeCaller, Mutect2, Strelka2, DRAGEN Germline, and DRAGEN Somatic pipelines. Among the approaches evaluated, FreeBayes most consistently represented simulated complex indels as single nearby variant records. We then developed a parsing workflow that derives effective deleted and inserted sequences from FreeBayes VCF output and enriches for candidate complex indels. This approach supports analysis of naturally occurring DSB repair events in single human colon crypts.

bioinformatics↗

bioRxiv2026

Fine Structural Features of Complex InDels and NHEJ Repair at Naturally Occurring Damage Sites in Normal Human Colon Crypts

DNA repair in biochemical and genetic experimental systems permits a precise definition of enzyme requirements and mechanistic steps. Comparing these findings to repair events at naturally occurring damage sites in multicellular organisms is essential for confirming and expanding these insights into a physiologic context. However, heterogeneity in any normal cell population increases with each cell division, and the reliable detection of replication-independent DNA damage sites and their repair has been a major barrier. Here, we examine single human colon crypts, which harbor natural cell clones, using a novel whole-genome sequencing (WGS) method to identify complex insertion-deletion (indel) in the crypt stem cells. Analysis of complex indel events likely repaired by non-homologous end joining occurring in crypt stem cells permits inferences about the in vivo repair of naturally occurring DNA damage within physiologically-relevant chromatin in normal human cells.

genomics↗

bioRxiv2025

High-depth Whole Genome Sequencing of Single Human Colon Crypts Uncovers New View on Crypt Clonality

It is generally accepted that each colon crypt is monoclonal and is populated by a single stem cell lineage after early human life. It has been impossible to profile somatic mutations genome-wide because high-depth and high-quality whole genome sequencing (WGS) of single cells is unachievable without tissue culture or whole genome amplification (WGA). Therefore, the cell-to-cell heterogeneity in each individual remains mostly unknown. Applying our novel WGA-free WGS method to obtain >30X post-alignment depth, we comprehensively profiled somatic mutations in 71 single human colon crypts with matched bulk controls from 14 individuals. Analysis reveals that colon crypts are commonly of multiple lineages in human adults. Our study is the first to demonstrate that an appropriately designed WGS approach can determine cell- to-cell heterogeneity in natural cell clones. The much higher sensitivity of WGS than the previous methods in lineage tracing can unlock the complex stem cell dynamics in the colon crypt.

genomics↗

bioRxiv2025

Analysis of Naturally Occurring Somatic Insertions in the Human Genome

Biochemical and genetic experimental systems permit precise definition of enzyme requirements and mechanistic steps in DNA repair. Comparison of these findings to repair events at naturally occurring breakage sites in multicellular organisms is valuable for confirming and extending these insights. However, heterogeneity in any cell population increases with each cell division, and the reliable detection of DNA breakage sites and their repair in vivo has been difficult due to technical limitations. Here, we examine somatic insertional mutations naturally occurring during normal metabolism and cell division in single human colon crypts using a novel whole-genome sequencing method. We find that replication slippage is a dominant mechanism for these events, and insertions larger than 10 bp are uncommon. Mechanistic features of these sites in physiologically normal cell clones, such as single human colon crypts, permits inferences about the DNA breakage repair zone and processing within natural chromatin, thereby permitting comparisons to experimental studies using ex vivo cellular and simplified biochemical systems.

genomics↗